1. Field of the Invention
The present application describes methods for separating sperm cells, sperm cell DNA, and other DNA from biological samples comprising both sperm cells and epithelial cells. The methods include selective digestion of epithelial cells and subsequent removal of sperm cells from the mixture by filtration through a nanofiber filter. DNA profiles obtained from the separated components of forensic samples are suitable for use in human identification.
2. Description of the Related Art
Forensic DNA testing has become widely used in sexual assault cases. However, because of the increasing use of DNA analysis and the lack of sufficient funding, a major backlog of cases waiting to be analyzed currently exists throughout the United States. The backlog of untested evidence in sexual assault cases is an important, controversial challenge facing our nation today. It is not uncommon for sexual assault kit (or “rape kit”) evidence to be stored for years before analysis, if analyzed at all. The backlog of rape kit evidence was estimated at 400,000 cases nationwide in 2014 (cnn.com, accessed Dec. 11, 2014: “400,000 rape kits left untested in U.S.”).
The reasons that large numbers of sexual assault kits are stored untested in police property rooms around the country are complex. Based on various circumstances, investigators or prosecutors may not have submitted them to a laboratory and requested that they be analyzed. Law enforcement agencies often lack the technology to track untested rape kits and the personnel needed for shipping or transporting untested kits to a crime lab in a timely manner. These agencies further lack resources and staffing to investigate and follow up on leads resulting from rape kit testing. Once in the forensic science laboratory, laboratory directors fault the time and cost requirements of these analyses as the bottleneck to DNA analysis (V. W. Weedn & J. W. Hicks, The unrealized potential of DNA testing, National Institute of Justice Journal, Issue 234 (December, 1997)). The most time-consuming step in DNA analysis of sexual assault evidence is the conventional differential extraction process that is used to separate sperm cells from other types of cells in a sexual assault sample (B. Budowle, et al., Simple protocols for typing forensic biological evidence: chemiluminescent detection for human DNA quantitation and restriction fragment length polymorphism (RFLP) analyses and manual typing of polymerase chain reaction (PCR) amplified polymorphisms, Electrophoresis 16(9): 1559-1567 (1995)). Therefore, the development of a quick, accurate, and effective method to streamline the differential extraction process would be advantageous in reducing, the rape kit backlog that exists in crime laboratories across the country today.
Forensic DNA analysis of sexual assault samples first requires extraction of the DNA in a differential manner to obtain separate male and female fractions of DNA from the sperm and epithelial cell donors, respectively. The female fraction can be used to verify the presence of the victim's DNA, and the male fraction to identify the perpetrator, or sperm donor. DNA extraction is typically followed by real-time polymerase chain reaction (PCR) quantitation, PCR amplification of genetic markers, separation of the PCR products, and data analysis. To date, efforts have been directed toward improving the speed and efficiency of sample processing for the latter steps, namely PCR (R. P. Oda, et al., Infrared-mediated thermocycling for ultrafirst polymerase chain reaction amplification of DNA, Anal. Chem. 70(20): 4361-4368 (1998); A. F. R. Huhmer & J. P. Landers, Noncontact infrared-mediated thermocycling for effective polymerase chain reaction amplification of DNA in nanoliter volumes, Anal. Chem. 72(21); 5507-5512 (2000); B. C. Giordano, et al., Polymerase chain reaction in polymeric microchips: DNA amplification in less than 240 seconds, Anal. Biochem. 291(1): 124-132 (2001); E. T. Lagally, et al., Single-molecule DNA amplification and analysis in an integrated microfluidic device, Anal. Chem. 73(3): 565-5μ(2001); E. T. Lagally, et al., Fully integrated PCR-capillary electrophoresis microsystem for DNA analysis, Lab on a Chip 1(2): 102-107 (2001)), DNA separation (D. Schmalzing, et al., DNA typing in thirty seconds with a microfabricated device, Proc. Nat. Acad. Sci. 94(19): 10273-10278 (1997); C. A. Emrich, et al., Microfabricated 384-lane capillary array electrophoresis bioanalyzer for ultrahigh-throughput genetic analysis, Anal. Chem. 74(19): 5076-5083 (2002)), and data analysis (M. W. Perlin, et al., Validating TrueAllele® DNA mixture interpretation, J. Forensic Sci. 56(6): 1430-1447 (2011)). The ability to reproducibly obtain clean sperm fraction DNA profiles from the differential extraction impacts the downstream processes, namely the time required for data analysis and interpretation is drastically less for a profile containing one donor versus one containing a mixture of donors. Sperm profiles containing a mixture of victim and perpetrator DNA oftentimes lead to difficult result interpretation, and this is currently a controversial topic in the forensic field (Brinkerhoff and Straehley, FBI Errors Lead to Discovery that DNA Evidence May be Far Less Foolproof When It includes More than One Person, http://www.allgov.com/news/unusual-news./fbi-errors-lead-to-discovery-that-dna-evidence-may-be-far-less-foolproof-when-it-includes-more-than-one-person-150910?news=857388) (accessed 2 Feb. 2016). However, little improvement has been made in the differential extraction process, which is the most important and most time-consuming step of the DNA analysis. In particular, robotic automation of the extensive differential extraction process has been shown to improve sample processing efficiency and throughput (12).
Sperm cell donor DNA is typically most easily obtained from sperm cells collected on vaginal swabs, taken in the routine collection of sexual assault evidence. The majority of genetic material collected on such swabs is from the victim (P. Wiegand, et al., DNA extraction from mixtures of body fluid using mild preferential lysis, Int. J. Legal Med. 104: 359-360 (1992)), mainly from epithelial cells that are collected from the vaginal lining. These cells, or DNA from these cells, must be separated from the sperm cells before sperm DNA is recovered and amplified for analysis by capillary electrophoresis. The standard process to isolate sperm cells from the victim's cells in forensic samples is performed in most crime laboratories by chemical means, involving differential lysis of the cells collected on the vaginal swab and exploiting the differential stability of the cell membranes reported in 1985 by P. Gill and coworkers (P. Gill, et al., Forensic application of DNA ‘fingerprints’, Nature 318(6046): 577-579 (1985); Qiagen Supplementary Protocol: Purification of DNA from epithelial cells mixed with sperm cells using the MagAttract® DNA Mini M48 kit. Qiagen (2010); K. Yoshida, et al., The modified method of two-step differential extraction of sperm and vaginal epithelial cell DNA from vaginal fluid mixed with semen, Forensic Sci. Int. 72(1): 25-33 (1995)). This multistep procedure begins by lysing the epithelial cells using mild conditions. The intact sperm cells (predominately heads because tails are often degraded) are pelleted by centrifugation, allowing the soluble DNA from the epithelial cells to be removed in the supernatant (this becomes known as the “epithelial” or “non-sperm” fraction). Several washing and centrifugation steps are involved in this process to remove non-sperm DNA from the sperm cells during which the analyst attempts to pipette as much of the supernatant (epithelial cell fraction) as possible from the tube with the sperm pellet without disturbing the sperm pellet. The several centrifugation and washing steps typically result in significant loss of valuable sperm cell evidence. The pelleted sperm cells are then suspended and lysed using a proteinase enzyme such as proteinase K in a buffer that contains detergent and a reducing agent such as dithiothreitol (DTT) for reduction of disulfide bonds. The DNA is extracted using phenol/chloroform/isoamyl alcohol or other DNA isolation methods. This process of partial digestion is commonly referred as the differential digest procedure. This differential extraction procedure is time consuming, not amenable to automation, and prone to loss of valuable crime scene evidence (Budowle, et al., cited above).
When the amount of epithelial cell DNA is very large and there are relatively few sperm cells, it is often difficult and sometimes impossible to remove enough of the epithelial DNA so that a clean sperm DNA profile is obtained, and the result oftentimes is a mixed DNA profile. Although this is the most common differential extraction method used by crime laboratories, it is very technique-dependent, and the quality of results can vary between analysts or technicians. The sperm pellet washing steps can be inefficient at removing soluble DNA from the cell pellet, leading to incomplete separation of sperm and non-sperm fractions, particularly in samples that contain large numbers of the victim's cells relative to sperm cells. In addition, the time-consuming nature of the process precludes this method as a viable solution in an efficiency-minded laboratory.
Over the last 30 years, several attempts have been made to address the inherent complexity and drawbacks of this method, attempting to improve ease of use, ability to automate and yield of recovered sperm DNA from forensic samples, particularly sexual assault kits. Following are the different approaches previously proposed to replace the centrifugation based differential digest protocol:
Density Medium Based Separation:
The separation is done by contacting the aqueous sample with a non-aqueous liquid having a density greater than about 1.00 g/cm3, wherein the density of the non-aqueous liquid is sufficiently low to permit pelleting of at least a portion of the sperm cells in the sample. A commercial kit is available from Promega Corporation (A. Tereba, et al., Methods and kits for isolating sperm cells, U.S. Pat. No. 7,320,891 B2, issued Jan. 22, 2008) and is in use in some laboratories. However, the process is still not easy to perform and sperm DNA separation/recovery can be variable and sub-optimal. Although it can be automated, this is much more complex than the manual method.
Immunological or Other Affinity Based Methods:
A reasonable alternative to the current method involves the separation of the sperm and epithelial cells before DNA extraction. Eisenberg (A. J. Eisenberg, Development of a spermatozoa capture system for the differential extraction of sexual assault evidence, presented at Profiling PCR and Beyond, Washington, D.C., Jun. 28, 2002) has addressed the separation of sperm and epithelial cells through the development of antibody-based separation schemes, using magnetic beads with covalently bound sperm-specific antibodies to selectively retain the sperm heads. There are potential problems associated with this approach, most notably clogging of the separation column by the large numbers of epithelial cells in casework samples. In addition to clogging, drawbacks of this technique include the cost of the materials required for the antibody/bead separation method, combined with the numerous steps requited to yield PCR-ready DNA.
A second method for the selection of sperm cells was reported by Elliott and coworkers (K. Elliott, et al., Use of laser microdissection greatly improves the recovery of DNA from sperm on microscope slides, Forensic Sci. Int. 137(1); 28-36 (2003)), who prepared slides from swabs and then selectively captured the sperm cells from the slide, using laser capture micro dissection. This method is also capable of isolating the sperm cells selectively; however, it is time-consuming, labor-intensive (to identify the sperm cells in the sample), and not likely to be amenable to high-throughput applications. In a similar process, Schoen and coworkers (W. M. Schoell, et al., Separation of sperm and vaginal cells with flow cytometry for DNA typing after sexual assault, Obstet. Gynecol. 94(4): (23-627 (1999)) demonstrated a fluorescence-activated cell sorting method for the separation of sperm and vaginal cells. However, the authors indicate that the use of this method would require altering the collection of evidentiary samples from vaginal swabs to vaginal lavages. Several other variations of the antibody based approach have been attempted and reported in the literature (G. Sanders, et al., Method for purification and identification of sperm cells, U.S. Pat. No. 8,703,416 B2, issued Apr. 22, 2014; Li, et al., Magnetic bead-based separation of sperm from buccal epithelial cells using a monoclonal antibody against MOSPD3, Int. J. Legal Med. 128: 905-911 (2014). An affinity binding approach using sperm binding protein was suggested by Sinha (S. K. Sinha, et al., Novel method for separation of human sperm from biological samples for application in human identification, U.S. Patent Application Pub. 2006/0141512 A1, published Jun. 29, 2006).
Microfluidic Based Methods:
The research group of Landers reported a microfluidic based platform to create an automated sperm separation apparatus (K. M. Horsman, et al., Separation of sperm and epithelial cells in a microfabricated device: potential application to forensic analysis of sexual assault evidence, Anal. Chem. 77(3): 742-749 (2005)). This separation utilizes the differential physical properties of the cells that result in settling of the epithelial cells to the bottom of the inlet reservoir and subsequent adherence to the glass substrate. As a result, low flow rates can be used to separate the sperm cells from the epithelial cell-containing biological mixture.
Another approach using a micro fabricated device used holographic optical trapping to sort objects and contaminants. This may be done using a chip for sorting utilizing holographic optical trapping (HOT), in the absence or presence of microfluidic streaming and sorting (T. Chakrabarty, Method and apparatus for sorting objects in forensic DNA analysis and medical diagnostics, U.S. Patent Application Pub. 2011/0223653 A1, published Sep. 15, 2011).
DNAse Digestion Based Method:
A promising method reported by Garvin and coworkers (A. M. Garvin, et al., DNA preparation from sexual assault cases by selective degradation of contaminating DNA from the victim, J. Forensic Sci. 54(6): 1297-1303 (2009)) is based on purifying sperm DNA from vaginal swabs taken from rape victims by selectively digesting the victim's epithelial cells to solubilize the victim's DNA, and to remove the soluble victim's DNA by selectively degrading it using, a nuclease, DNase I. Although the method works (A. M. Garvin, et al., Isolating DNA from sexual assault cases: a comparison of standard methods with a nuclease-based approach, Investigative Genetics 3: 25 (2012)), the fear of introducing a DNA degrading enzyme, in a valuable forensic DNA sample, is not appealing to forensic scientists.
Selectively Lysing the Sperm Cells:
In an approach opposite to the commonly used method whereby the non-sperm cells are lysed and intact sperm cells are recovered by centrifugation (P. Gill, et al., cited above), this method selectively lyses the sperm cells in presence of other cell types and collects the sperm DNA. The sperm cell lysis is effected by chemical reagents such as DTT in buffer (J. Y. Liu, Method for recovering sperm nucleic acid from a forensic sample, U.S. Patent Application Pub. No 2009/0042255 A1, published Feb. 12, 2009).
Physical Separation by Filtration Based Methods:
Chen and coworkers (J. Chen, et al., A physical method for separating spermatozoa from epithelial cells in sexual assault evidence, J. Forensic Sci. 43(1): 114-118 (1 998)) demonstrated a separation of sperm cells from a mixture of sperm and epithelial cells using an 8-μm nylon mesh membrane filter, which retains the larger epithelial cells but allows the sperm cells to pass through it. In this method about 70% of the smaller sperm cells will pass through the filter whereas only 1-2% of the larger epithelial cells will pass through the membrane filter. However, the presence of female DNA in the sperm cell fraction is a factor in this method, because epithelial cells easily lyse, allowing free DNA or epithelial cell nuclei to in pass through the nylon mesh filter. Following the separation in all of these techniques, the purified sperm undergo normal forensic DNA analysis for genetic identification.
Another filtration method proposed by Garvin reports separation of sperm and non-sperm cells utilizing a track-etched filter or a laser track etched filter, or a combination of the two. The track etched filter typically has a mean pore size of about 2 μm. This method is similar to the method reported by Chen (Id.), but instead of nylon filters uses a track etched filter of defined pore size (A. M. Garvin, Filtration based DNA preparation for sexual assault cases, J. Forensic Sci. 48(5): 1084-1087 (2003)).
Description of Polymers Useful in the Invention:
Nanofibers are often defined as fibers with diameters of less than 100 nanometers (1×10−7 m). However, for the present purpose, nanofibers having diameters as large as about 700 nm may be useful. Nanofibers can be produced by interfacial polymerization, electrospinning or electrostatic spinning, melt-blowing, bicomponent fiber spinning, phase separation, template synthesis, or self-assembly using a variety of materials. Nanofibers can be produced from many synthetic polymers such as vinyl polymers, acrylic polymers, polyesters, polyethers, polycarbonate and polyimides. Many other types of polymer and copolymers using several biocompatible polymers such as polycaprolactone have been reported to be used to produce nanofibers (H. Fong & D. H. Reneker, in Structure formation in polymeric fibers: Chapter 6, Electrospinning and formation of nanofibers, D. R. Salem, ed., Munich: Carl Hanser Verlag, pp. 225-246, 2001 (ISBN: 3-446-18203-9); T. J. Menkhaus, et al., Applications of electrospun nanofiber membranes for bioseparations, Hauppauge, N.Y.: Nova Science Publishers, 2010 (ISBN: 978-1608767823); Z.-M. Huang, et al., A review on polymer nanofibers by electrospinning and their applications in nanocomposites, Composites Science and Technology 63: 2223-2253 (2003)).
The electrospinning process provides the ability to produce nanofibers from various materials in large quantity with specific application based properties. Recently, nanofiber membranes have been used for many bio-separation applications (31). Nanofibers can be modified with specific functional groups and specific biomolecules can be covalently attached for affinity separations. Many biomolecules such as proteins, DNA, and pathogens can be captured on certain nanofibers using adsorption processes.
Another way of producing nanofibers is by centrifugal spinning, which allows production of a variety of materials at high speed and low cost (Taghavi and Larson, Regularized thin-fiber model for nanofiber formation by centrifugal spinning, Phys. Rev. E 89 and Zhang and Lu, Centrifugal Spinning: An Alternative Approach to Fabricate Nanofibers at High Speed and Low Cost, Polymer Reviews Volume 54, Issue 4, 2014). In centrifugal spinning, the spinning fluid is placed in a rotating spinning head. When the rotating speed reaches a critical value, the centrifugal force overcomes the surface tension of the spinning fluid to eject a liquid jet from the nozzle tip of the spinning head. The jet then undergoes a stretching process and is eventually deposited on the collector, forming solidified nanofibers. Centrifugal spinning is simple and enables the rapid fabrication of nanofibers for various applications. Nanofibers composed of polypropylene, polyvinylidine fluoride, or polybutylene terephthalate can be produced with this method.
Separation with Nanofiber Membranes:
Separations with nanofiber membranes can be accomplished by using nanofiber layers of selected apparent pore size as a filtration membrane. Hollow nanofiber tube bundles can also be used by aligning the flow parallel to the nanofiber tube. Size based separations are routinely used for bioprocessing. Depth filtration and microfiltration are common operations used for clarification of fermentation broth where cells of approximately 20-200 nm and cellular debris 0.1-1 μm are removed from bioreactors (D. J. Roush & Y. Lu, Advances in primary recovery: centrifugation and membrane technology, Biotechnology Progress 24(3): 488-49 (2008)). Nanofiltration with membranes is used for viral clearance and/or purification of 20-200 nm virus particles, and ultrafiltration is commonly used for purification or separation of proteins (R. van Reis & A. Zydney, Membrane separations in biotechnology, Current Opinion in Biotechnology 12(2): 208-211 (2001)). In these cases, well defined pore size and size cutoff is needed to make effective separation. Also high porosity of materials is needed to avoid fouling and subsequent clogging of the membrane. Another important characteristic required when separating biomolecules with nanofiltration is chemical and physical robustness. Nanofiber felts can be produced from mechanically and chemically strong fibers with well controlled pore size among fibers or can be produced as collections of hollow fibers. These nanofiber production features offer a unique opportunity for using them as a size based separation medium for specific bio-particles or cells such as sperm cells. Polymer, carbon and ceramic nanofibers are also able to separate with high flux (R. S. Barhate, et al., Preparation and characterization of nanofibrous filtering media, J. Membrane Sci. 283(1-2): 209-218 (2006)). Polymer nanofibers show in general the least amount nonspecific binding of molecules such as proteins or DNA but may suffer from less chemical robustness than the ceramic fibers. Ceramic fibers suffer from being brittle and have a potential for non-specific adsorption of bio-particles or bin-molecules resulting in fouling. However, ceramic nanofibers can withstand harsh chemical regeneration processes.
Electrospun carbon nanofibers are made from the electrospun nanofibers including polyacrylonitrile (PAN) pitch cellulose, and polyvinyl alcohol (PVA) nanofibers. Carbon nanofibers made from PAN nanofibers possess very ordered graphite crystalline structure. Pore sizes among the nanofibers can be controlled in the range from tens to hundreds of nanometers which makes the nanofiber capable of filtering contaminants with sizes larger than the apparent pore sizes of the nanofiber fabrics. This gives the nanofiber the ability to filter bacteria and viruses. Carbon nanofibers made of PVA possess a large amount of nano-sized pores (H. Fong & D. H. Reneker, cited above). Another very interesting proper y of nanofiber is the ability to produce hollow nanofibers (D. Li & Y. Xia, Direct fabrication of composite and ceramic hollow nanofibers by electrospinning, Nano Letters 4(5): 933-938 (2004)). Such hollow nanofiber bundles can be used for size based bio-separations with liquid flowing parallel to the fiber tube (C. A. Grimes, Synthesis and application of highly ordered arrays of TiO2nanotubes, J. Materials Chem. 17(15): 1451-1457 (2007)).